title: Template-directed fluorogenic oligonucleotide ligation using "click" chemistry: detection of single nucleotide polymorphism in the human p53 tumor suppressor gene.
abstract: A novel nonfluorescent alkyne-modified coumarin phosphoramidite was synthesized and successfully incorporated into oligonucleotides, which were then used in highly efficient DNA interstrand cross-linking and ligation reactions via "click" chemistry. The template-directed fluorogenic ligation "click" chemistry reaction was used for single nucleotide polymorphism analysis, where the target DNA catalyzes the ligation of two nonfluorescent probes to generate a fluorescent product. The upstream oligonucleotide probe is a nonfluorescent alkyne-modified coumarin and the downstream probe is an azide-modified oligonucleotide. When bound to a fully complementary template, the oligonucleotides ligated to produce a fluorescent product with a fluorophore at the ligation point. Wild-type and mutant p53 alleles were used to demonstrate that template-directed fluorogenic oligonucleotide ligation is sequence-specific and is capable of single nucleotide discrimination under mild conditions, even without the removal of unreacted probes.
authors.affiliation: Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 North Cramer Street, Milwaukee, WI 53211, USA.
authors.forename: Huabing
authors.initials: H
authors.lastname: Sun
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Peng
mesh_terms: D001483:Base Sequence; D005456:Fluorescent Dyes / Q000737:chemistry*; D016158:Genes, p53; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009841:Oligonucleotides / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D013698:Templates, Genetic
chemical_list: D005456:Fluorescent Dyes; D009841:Oligonucleotides